In vivo selective expression of thyroid hormone receptor α1 in endothelial cells attenuates myocardial injury in experimental myocardial infarction in mice - PubMed (original) (raw)

In vivo selective expression of thyroid hormone receptor α1 in endothelial cells attenuates myocardial injury in experimental myocardial infarction in mice

Jorge Suarez et al. Am J Physiol Regul Integr Comp Physiol. 2014.

Abstract

Ischemic heart disease (IHD) is the single most common cause of death. New approaches to enhance myocardial perfusion are needed to improve outcomes for patients with IHD. Thyroid hormones (TH) are known to increase blood flow; however, their usefulness for increasing perfusion in IHD is limited because TH accelerates heart rate, which can be detrimental. Therefore, selective activation of TH effects is desirable. We hypothesized that cell-type-specific TH receptor (TR) expression can increase TH action in the heart, while avoiding the negative consequences of TH treatment. We generated a binary transgenic (BTG) mouse that selectively expresses TRα1 in endothelial cells in a tetracycline-inducible fashion. In BTG mice, endothelial TRα1 protein expression was increased by twofold, which, in turn, increased coronary blood flow by 77%, coronary conductance by 60%, and coronary reserve by 47% compared with wild-type mice. Systemic blood pressure was decreased by 20% in BTG mice after TRα1 expression. No effects on heart rate were observed. Endothelial TRα1 expression activated AKT/endothelial nitric oxide synthase pathway and increased A2AR adenosine receptor. Furthermore, hearts from BTG mice overexpressing TRα1 that were submitted to 20 min ischemia and 20 min reperfusion showed a 20% decline in left ventricular pressure (LVP) compared with control mice where LVP was decreased by 42%. Studies using an infarction mouse model demonstrated that endothelial overexpression of TRα1 decreased infarct size by 45%. In conclusion, selective expression of TRα1 in endothelial cells protects the heart against injury after an ischemic insult and does not result in adverse cardiac or systemic effects.

Keywords: coronary circulation; endothelial; receptor α1; thyroid hormones; transgenic.

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Figures

Fig. 1.

Tie2-Tet-TRα1 Binary transgenic mice (BTG) expresses thyroid hormone receptor α1 (TRα1) in endothelial cells in a Tet-inducible fashion. A: transgenic mouse constructs. B: TRα1 transgene-encoded mRNA was found only in the endothelial cells (ECs) from BTG mice given doxycycline (Dox). mRNA was reverse transcribed, and RT-PCR was performed using primers specific for the transgenic TRα1 transcript. 18s rRNA was used as an endogenous control. Lane 1: cardiac myocytes (CMs) from BTG mice given Dox; lane 2: CMs from control mice; lane 3: ECs from BTG mice given Dox; and lane 4: ECs from control mice. C: twofold Dox-mediated increase in TRα1 transgene protein expression was seen in coronary derived ECs, but not in CMs. *P = 0.006; n = 3 for each line. Each sample corresponds to cells from two hearts.

Fig. 2.

Influence of TRα1 expression on coronary circulation. TRα1 expression in endothelial cells increased coronary blood flow (A) (*P = 0.040; n = 3), coronary conductance (B) (*P = 0.0213; n = 4) and coronary reserve (C) (*P = 0.024; n = 5) vs. WT + Dox. D: effect of T3 on coronary reserve in wild-type (WT) mice and mice with endothelial TRα1 expression (BTG) (n = 5).

Fig. 3.

Potential undesirable effects in BTG + Dox mice. A: heart rate. B: body temperature. C: arterial blood pressure. D: cardiac O2 consumption. Endothelial expression of TRα1 did not alter heart rate or body temperature. Blood pressure is 20% lower in BTG + Dox compared with WT + Dox (*P = 0.038; n = 5). Oxygen consumption increased to a lesser extent in BTG + Dox mice compared with WT + Dox mice as the rate pressure product increased (#P < 0.001; n = 5).

Fig. 4.

Endothelial TRα1 expression activates the AKT/endothelial nitric oxide synthase (eNOS) signaling pathway. A: phosphorylated AKT is increased in endothelial cells from BTG + Dox mice. Phosphorylated eNOS is also increased by TRα1 expression (*P < 0.05; n = 3, each sample is a pool of cells from two mouse hearts). B: influence of thyroid hormone receptor (TR) expression on eNOS. Rat aortic endothelial cells in culture were infected with adenovirus expressing TRα1 (Adv.TRα), TRβ1 (Adv.TRβ), or with an empty vector control adenovirus (Adv.Ctr). Western blots show that eNOS expression was not changed by TR expression. Phosphorylated eNOS (P-eNOS) was increased by 63% with TRα1 expression. TRβ1 expression did not change P-eNOS. The P-eNOS/eNOS ratio followed the same trend. Bar graphs show summarized data of densitometry of the bands. *P < 0.05 vs. Adv.Ctr.; n = 6 for each group from three separate experiments. Adv.TRβ and Adv.Ctr were not statistically different.

Fig. 5.

A: TRα1 expression inhibits MAPK/ERK activation, but increases A2a adenosine receptor expression in endothelial cells. 1: Western blot showing total and phosphorylated MAPK and adenosine 2A receptors (A2AR) levels. 2: bar graphs show summarized data of densitometric analysis of three samples per group. Each sample is a pool of cells from two mice *P < 0.05 vs. control. B: TRα1 expression has no significant effect on capillary density in the heart. Graph represents averaged data (means ± SE) showing the capillary density in WT + Dox and BTG + Dox hearts. No statistically significant differences were observed; n = 3 for each group.

Fig. 6.

Influence of endothelial TRα1 expression on ischemic insult. A: TRα1 protein level is reduced after ischemia/reperfusion (I/R) in endothelial cells obtained from isolated mouse hearts. B: similar results were observed in simulated (Sim) I/R in endothelial cells in culture. C: beneficial effects of endothelial TRα1 expression on I/R-mediated cardiac injury. Developed pressure in the left ventricle (LVP) after 20 min of no-flow ischemia. In Wt + Dox mice LVP decreased by 40% vs. only a 20% decline in BTG + Dox mice. *P < 0.05 vs. WT; &P < 0.05 vs. WT I/R; n = 5 for each group. D: effect of endothelial TRα1 overexpression on myocardial infarct (MI) size. In BTG + Dox mice in vivo MI size (MIS) is decreased by 23% determined as area at risk (AAR) and by 31% as MIS over left ventricle mass (LV). *P < 0.05 vs. WT; n = 4 for each group.

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In vivo selective expression of thyroid hormone receptor α1 in endothelial cells attenuates myocardial injury in experimental myocardial infarction in mice - PubMed (original) (raw)